Project Abstract: Rhabdomyosarcoma is a highly metastatic soft tissue malignancy of childhood for which new therapies are desperately needed. The clinical management of RMS patients has been largely unchanged over the past three decades, and is currently limited to surgical resection, radiotherapy, and combination chemotherapy. In this context, a mechanism-based targeted therapy would have potential for a transformative impact on RMS patient outcomes. The most common genetic event in RMS pathogenesis is a chromosomal rearrangement that produces the PAX-fusion oncoprotein, which is a chimeric transcription factor that deregulates chromatin and transcription to promote transformation. Our domain-focused CRISPR screens validate that RMS tumors retain a powerful addiction to the PAX-fusion, yet strategies for direct or indirect targeting of this ?undruggable? protein have yet to be successful. One obstacle in this endeavor is our incomplete understanding of the upstream and downstream factors that support the function of the PAX-fusion, which we seek to address with the research proposed here. Through deep molecular profiling of RMS cell lines depleted of the PAX-fusion, we have recently developed reporters which are compatible with flow cytometry-based measurements and cell sorting. This now allows us to perform saturating genetic screens to delineate all components of the PAX-fusion pathway in this disease. In the first aim of this study, we will perform CRISPR exon-scanning of the endogenous PAX-fusion locus, which is an assay we previously developed for exposing functionally important domains of cancer maintenance genes. These experiments will define the critical subregions of the fusion oncoprotein that deregulate transcription to sustain the block in myo-differentiation. The second aim of this proposal will leverage our recently developed paralog domain co-targeting methodology to expose all of the critical genes, and redundant paralogous gene pairs, that are critical for the PAX-fusion to carry out its function. The final aim of this project will identify the critical E3 ligase that acts to restrain PAX-fusion expression in RMS cells, whose function could be stimulated to degrade this oncoprotein. This two-year research project will employ the latest innovations in CRISPR-based genetic screening to establish an important resource for the RMS field; a genetic foundation for mechanism-based research of the PAX-fusion oncoprotein that will enable its pharmacological modulation with therapeutic intent.